The nucleotide sequence of the human cytomegalovirus replication origin of strain Towne (an AatII-SacI fragment corresponding to nt 90372-94637 of strain AD169) was determined and compared with AD169. Two differences were found in the nucleotide sequence level. One was the alteration of structural organization (a major difference): a 189-bp region of AD169 (nt 93337-93525) was directly repeated three times in Towne. The other was a change in the nucleotide residue level including substitution, insertion, or deletion (a minor difference). The divergent residues were predominantly localized within the nt 92591-92855 region of AD169. A replication assay revealed that replication ability remained after deletion of the 189-bp repeat but disappeared after either a 1.5-kb deletion from the AatII end or a 0.9-kb deletion from the SacI end. The 1.5- and 0.9-kb regions were relatively conserved. These results indicate that at least two regions essential for replication ability lie outside of both the relatively variable region and the 189-bp repeat and suggest that these essential regions support replication even with a spatial separation of either one (AD169) or three repeats (Towne) of the 189-bp region.